Antibacterial Evaluation of Sophoraflavanone G from Sophora pachycarpa Against Pseudomonas aeruginosa and Staphylococcus epidermidis Biofilm

Antibacterial Evaluation of Sophoraflavanone G from Sophora pachycarpa Against Pseudomonas aeruginosa and Staphylococcus epidermidis Biofilm | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antibacterial Evaluation of Sophoraflavanone G from Sophora pachycarpa Against Pseudomonas aeruginosa and Staphylococcus epidermidis Biofilm Niloofar Zanganeh, Ghazaleh Hatamian, Abolfazl Shakeri, Bibi Sedigheh Fazly Bazzaz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7299693/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Antimicrobial resistance, particularly among biofilm-forming pathogens, poses a serious threat to public health and drives the urgent need for new therapeutic agents. Sophoraflavanone G, a prenylated flavonoid derived from Sophora pachycarpa , has shown notable antimicrobial activity, especially against Gram-positive bacteria. In this study, Sophoraflavanone G was isolated and purified using chromatographic techniques and structurally confirmed via ¹H-NMR. Its antibacterial and anti-biofilm properties were evaluated against several bacterial strains, including Pseudomonas aeruginosa PAO1 and Staphylococcus epidermidis DSMZ 3270. The compound exhibited potent inhibitory and bactericidal activity against all tested Gram-positive bacteria, with Listeria monocytogenes being the most sensitive (MIC = 0.98 µg/mL). However, it showed no activity against P. aeruginosa in planktonic form (MIC > 1000 µg/mL). While it failed to inhibit P. aeruginosa biofilm formation or enhance tobramycin penetration at low doses, a higher concentration (1 mg/mL) of Sophoraflavanone G significantly improved antibiotic penetration into the biofilm. In contrast, the compound demonstrated strong inhibitory, disruptive, and biofilm-penetrating effects against S. epidermidis , with a clear dose-dependent response. These findings underscore the potential of Sophoraflavanone G as a candidate for managing Gram-positive and biofilm-associated infections, particularly those involving S. epidermidis , while highlighting the need for further development to improve its efficacy against Gram-negative pathogens. Sophoraflavanone G Biofilm Pseudomonas aeruginosa Staphylococcus epidermidis Antimicrobial activity Plant-derived compound Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Antibiotic resistance (AMR) is a critical global public health issue that threatens the effectiveness of antibiotics in treating bacterial infections. This phenomenon is not only a modern challenge but has ancient origins, as bacteria have evolved resistance mechanisms over millions of years [ 1 ]. The widespread use of antibiotics in human medicine, veterinary practice, and agriculture has accelerated the development and spread of resistant bacteria, creating a complex web of resistance elements that are genetically diverse and mechanistically sophisticated [ 1 , 2 ]. Biofilm-forming bacteria play a significant role in persistent infections due to their unique structural and functional characteristics that enhance survival against antimicrobial treatments and host defenses. These bacteria, such as Pseudomonas aeruginosa , form complex communities encased in an extracellular polymeric substance (EPS) matrix, which not only protects them from antibiotics but also facilitates genetic exchange, including antibiotic resistance genes [ 3 , 4 ]. Moreover, the EPS matrix acts as a physical barrier, creating gradients of nutrients and oxygen that contribute to the metabolic dormancy of some cells, making them less susceptible to antimicrobial agents [ 3 , 5 ]. Additionally, biofilms can impair the activation of immune responses, such as phagocytosis and the complement system, allowing bacteria to evade host defenses [ 6 , 7 ]. P. aeruginosa PAO1 is a significant pathogen in clinical settings due to its robust antibiotic resistance mechanisms and ability to cause persistent infections. This bacterium is particularly problematic in hospital environments, where it contributes to nosocomial infections, especially in immunocompromised and cystic fibrosis patients. The resistance of P. aeruginosa to antibiotics is multifaceted, involving intrinsic mechanisms such as efflux pumps and acquired resistance through gene mutations and adaptive responses [ 8 ]. [ 9 ]. Therefore, innovative treatment strategies are being explored, including non-antibiotic approaches like quorum sensing inhibition, phage therapy, and nanoparticle-based treatments, although these approaches face challenges related to cost and safety [ 10 ]. Quorum sensing (QS) in P. aeruginosa plays a critical role in regulating virulence and biofilm formation, significantly contributing to its pathogenicity. This process involves the production of signaling molecules that coordinate gene expression related to virulence factors and biofilm development, enabling the bacteria to adapt and thrive in hostile environments, such as during infections in immunocompromised patients [ 16 , 17 ]. Disrupting QS can effectively reduce the pathogenicity of P. aeruginosa without exerting additional selective pressure for antibiotic resistance, as QS inhibitors (QSIs) can attenuate virulence without inhibiting bacterial growth [ 17 , 18 ]. Staphylococcus epidermidis , a common skin commensal, has emerged as a significant opportunistic pathogen, particularly in healthcare settings, due to its ability to cause infections such as septicaemia and endocarditis. The prevalence of antibiotic resistance among S. epidermidis strains is alarming, with studies indicating high resistance rates against penicillin (95.65%), tetracycline (91.30%), and methicillin (92.2%) among hospital isolates [ 11 , 12 ]. The bacterium's capacity to form biofilms enhances its virulence and resistance to antibiotic treatment, complicating management strategies [ 13 ]. Furthermore, the presence of resistance genes, such as mecA and various efflux pumps, contributes to its multi-drug resistant phenotype, making infections difficult to treat [ 14 ]. This multidrug resistance poses significant challenges for treatment, particularly in immunocompromised patients, highlighting the need for ongoing surveillance and novel therapeutic strategies [ 15 ]. In S. epidermidis , QS is a critical mechanism that regulates biofilm formation and virulence, primarily through the accessory gene regulator (AGR) system. This system is activated by autoinducing peptides (AIPs) that bind to the AgrC receptor, leading to the expression of virulence factors and biofilm dispersal agents such as phenol-soluble modulins [ 16 ]. Inhibiting QS in S. epidermidis is a promising strategy to combat biofilm-associated infections, particularly those involving drug-resistant strains. Various approaches have been explored, including the use of synthetic peptides and natural compounds [ 17 ]. Conventional antibiotics are often ineffective against biofilms due to several inherent challenges. Biofilms, which are communities of bacteria encased in a protective matrix, exhibit reduced susceptibility to antibiotics as these drugs typically target planctonic cells, while many cells within biofilms are metabolically inactive or in a persister state. In addition, the resistance is amplified by efflux pumps and physiological changes [ 18 , 19 ]. The exploration of plant-derived compounds as alternative antimicrobial agents has gained significant traction due to the rising threat of AMR and the limitations of conventional antibiotics. Various studies highlight the diverse antimicrobial properties of these compounds, which include essential oils, extracts, and bioactive substances from both medicinal and non-medicinal plants, as well as marine sources [ 20 – 22 ]. These plant-derived agents exhibit multi-target mechanisms, making them less prone to resistance compared to traditional single-target antibiotics [ 20 ]. Additionally, they have shown efficacy against biofilms, which are notoriously difficult to treat due to their protective matrices [ 23 ]. Sophora pachycarpa , a member of the Fabaceae family, has been utilized in traditional medicine for its diverse therapeutic properties, primarily attributed to its rich content of flavonoids, particularly prenylated flavonoids. Research has identified several bioactive compounds, including Sophoraflavanone G and Sophoraisoflavanone A, which exhibit significant biological activities such as anti-inflammatory, antibacterial, and cytotoxic effects [ 24 , 25 ]. Sophoraflavanone G, a prenylated flavonoid predominantly isolated from plants of the genus Sophora , has garnered attention for its notable antimicrobial properties. Recent studies have demonstrated its efficacy against a range of bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium [ 26 , 27 ]. Its mode of action is believed to involve disruption of bacterial cell membrane integrity and interference with peptidoglycan synthesis, making it a promising candidate for novel antimicrobial therapies [ 28 ]. Despite growing evidence supporting the broad-spectrum antibacterial activity of Sophoraflavanone G, its specific effects on biofilm formation and eradication remain inadequately characterizedand the potential of Sophoraflavanone G to act synergistically with conventional antibiotics, particularly tobramycin against P. aeruginosa has not been systematically evaluated. These knowledge gaps underscore the necessity for targeted investigations into the anti-biofilm and combinatory antibacterial properties of this compound, especially in the context of biofilm-mediated antimicrobial resistance. This study, tries to introduce the ability of Sophoraflavanone G in inhibition and disruption of biofilm, in both Gram-positive and Gram-negative bacteria, including P. aeruginosa and S. epidermidis , as well as its capacity to potentiate the effect of antibiotics. Material and Method Chemicals and Materials All solvents and reagents used in this study were of analytical grade. Silica gel GF254 plates and silica gel (230–400 mesh) for column chromatography were obtained from Merck (Germany). Ethyl acetate, petroleum ether (60–80°C), dichloromethane, methanol, and acetone were purchased from Dr. Mojallali Co. (Iran). Vanillin, sulfuric acid, sodium chloride (NaCl), glucose, crystal violet, and 2,3,5-triphenyl tetrazolium chloride (TTC) were also sourced from Merck (Germany). Dimethyl sulfoxide (DMSO) was obtained from Merck (Germany), and absolute ethanol was purchased from Simin Tak (Iran). Tryptic Soy Agar (TSA) was acquired from Himedia (India), while Mueller-Hinton Broth (MHB) was provided by Liofilchem (Italy). Tobramycin was sourced from Sigma (USA), and cloxacillin was obtained from Farabi (Iran). The bacterial strains used in this study included Pseudomonas aeruginosa PAO1 (Wild type Nottingham), Staphylococcus epidermidis (DSMZ 3270, Germany), Listeria monocytogenes (PTCC 1298, Iran), Bacillus subtilis (PTCC 1247, Iran), methicillin-sensitive Staphylococcus aureus (MSSA, PTCC 1431, Iran), methicillin-resistant S. aureus (MRSA, PTCC 1112, Iran), and Micrococcus luteus (PTCC 1110, Iran). All strains were maintained and handled according to standard microbiological practices. Extraction and Purification The roots of S. pachycarpa were collected in June 2019 from the Kalat region, Razavi Khorasan Province, Iran. Botanical identification was performed by Mrs. Soozani, and a voucher specimen (No. 13275) was deposited in the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. The air-dried roots (1350 g) were powdered using an electric grinder and macerated in 4 L of acetone for 24 hours at room temperature. The extract was filtered and concentrated under reduced pressure using a rotary evaporator to afford a reddish-brown residue (35.5 g). Isolation and Purification of Sophoraflavanone G and Structure Elucidation The crude extract was subjected to column chromatography on silica gel using a polarity-gradient elution system comprising petroleum ether, ethyl acetate, and methanol. A total of 65 fractions (200 mL each) were collected and concentrated under reduced pressure. The solvent ratios used for each fraction are listed in Table 5. Further purification of the target compound was achieved by preparative thin-layer chromatography (PTLC). Development was carried out in a petroleum ether:ethyl acetate (6:4) system. The resolved band corresponding to the standard was visualized under UV light, marked, scraped off, and extracted with acetone. The extract was filtered and the solvent was removed under reduced pressure to obtain the purified compound. The structure of compound was confirmed through spectral interpretation (1H-NMR) and comparison with previously published data [ 29 ]. Determination of MIC and MBC The MIC (Minimum Inhibitory Concentration) of sophoraflavanone G was tested against S. aureus , S. epidermidis , B. subtilis , L. monocytogenes , M. luteus , and P. aeruginosa PAO1. A 2 mg sample of the compound was dissolved in 70 µl DMSO (3.5%) and 930 µl MHB (Mueller-Hinton Broth). Serial dilutions (1000 − 0.49 µg/ml) were prepared in a 96-well plate, with each concentration tested in duplicate. Positive control wells contained only separate bacteria in MHB; negative controls contained no bacteria. After adding 20 µl bacterial suspension (10⁶ CFU/ml), the plates were incubated for 24 h. Then, 20 µl TTC (5 mg/ml) was added and incubated for another 1 h. The lowest concentration with no color change was reported as MIC. Following MIC assays, samples from wells with no red color were streaked on TSA agar and incubated for 24 h. The lowest concentration with no bacterial growth was reported as MBC (Minimum Bactericidal Concentration). Biofilm Assays Involving P. aeruginosa PAO1 To investigate the inhibitory effect of sophoraflavanone G on biofilm formation by P. aeruginosa PAO1, a suspension containing 10⁶ CFU/mL of the bacterium was prepared. A total of 20 µL of this suspension was added to 180 µL of glucose-enriched Mueller-Hinton broth (MHB) containing sophoraflavanone G at concentrations corresponding to ½ MIC (0.5 mg/mL). For the positive control, 180 µL of medium and 20 µL of bacterial suspension were used. The negative control wells contained only the medium. Plates were incubated at 37°C to allow biofilm formation. After 24 and 36 hours, the content of each well was gently aspirated and refreshed with medium containing the same concentration of sophoraflavanone G. At 48 hours, the wells were emptied and washed twice with 200 µL of normal saline. Subsequently, 50 µL of 0.03% crystal violet solution was added to each well and allowed to incubate for 10 minutes. Excess dye was then removed, and wells were washed at least twice to remove unbound stain. After drying, 200 µL of 95% ethanol was added to each well to solubilize the bound dye, and absorbance was measured at 590 nm using an ELISA reader to quantify the biomass of the formed biofilm. To investigate whether sophoraflavonone G facilitates the penetration of tobramycin into the constructed biofilm, first, biofilms were allowed to develop by adding 20 µL of bacterial suspension (10⁶ CFU/mL) to 180 µL of glucose-supplemented MHB in 96-well plates, followed by a 24-hour incubation. After this period, the medium was removed and replaced with fresh medium. At 36 hours, the wells were treated with either 1 mg/mL or 0.5 mg/mL of sophoraflavanone G in combination with tobramycin at its MIC level (2 µg/mL). At the 48-hour mark, the wells were washed and then received 180 µL of enriched medium and 20 µL of 5 mg/mL tetrazolium salt (TTC). Following a 4-hour incubation, absorbance at 450 nm was measured to determine metabolic activity, reflecting penetration and antibacterial activity within the biofilm. Wells treated with tobramycin only were used as control (2 µg/mL). The results compared with the positive controls which received no treatment. Biofilm Assays Involving S. epidermidis DSMZ 3270 In evaluating the ability of sophoraflavanone G to inhibit biofilm formation by S. epidermidis , a suspension containing 10⁶ CFU/mL was added (20 µL) to 180 µL of glucose-enriched MHB containing sophoraflavanone G at ½ MIC (3.9 µg/mL) and MIC (7.8 µg/mL). The plates were incubated, and after 24, and 36 hours, the medium was gently replaced with fresh medium of identical composition, preserving any forming biofilm. After 48 hours, the wells were washed and stained with crystal violet, followed by measurement of absorbance at 590 nm. In evaluating biofilm disruption, 20 µL of the bacterial suspension was combined with 180 µL of glucose-enriched MHB in 96-well plates. After 24-hours incubation, the medium was replaced, and biofilms were allowed to continue forming. At 36 hours, the wells were treated with sophoraflavanone G at ½ MIC and MIC concentrations. Positive controls received only fresh medium. After 48 hours, standard crystal violet staining was carried out and absorbance was measured at 590 nm. For determining compound penetration into established biofilms, the same procedure was followed, with treatments administered with the similar concentrations of sophoraflavanone G, after 36 hours. Again, at 48 hours, all wells were emptied and received 180 µL of fresh glucose-enriched MHB along with 20 µL of 5 mg/mL TTC solution. After a 4-hour incubation period, absorbance was read at 450 nm to assess bacterial metabolic activity within the biofilm matrix, serving as an indirect indicator of drug penetration. Statistical Analysis The results of each experiment were evaluated after three repetitions as the means ± standard deviation. All data were analyzed by SPSS software (version 27.0.1) using one-way ANOVA (Tamhane/Tukey's Multiple Comparison Tests) at significant level of p-value < 0.05. Ethics approval and consent to participate This article does not contain any studies with human participants or animals. All the plant species were collected from various locations by Dr. Milad Iranshahy following proper guidelines and legislation procedures and identified by Ms. Souzani (MSc of Systematic Botany, Department of Pharmacognosy, School of Pharmacy) and have been deposited at the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences, Iran. No specific license was required for the collection of the plants. Results and Discussion Sophoraflavanone G characterization Sophoraflavanone G, is a bioactive compound that exhibits significant antibacterial properties, particularly against E. faecium , by disrupting the bacterial cell wall. This disruption is evidenced by the compound's ability to bind to peptidoglycans, leading to cell wall damage and subsequent cell lysis, as confirmed by transmission electron microscopy [26]. Additionally, Sophoraflavanone G demonstrates synergistic antibacterial effects when combined with other antimicrobial agents, such as ampicillin and gentamicin, against various oral bacteria, significantly reducing the minimum inhibitory and bactericidal concentrations required for efficacy [30, 31]. Despite its promising pharmacological activities, including anti-inflammatory and antiproliferative effects, SFG's clinical application is limited by its poor water solubility and bioavailability, necessitating further research to enhance its therapeutic potential [32]. In this study, Sophoraflavanone G was isolated and purified using Silica gel column chromatography and preparative thin-layer chromatography (PTLC) using a petroleum ether:ethyl acetate system at a 6:4 ratio. The chemical structure of the compound is shown in Fig. 1. Based on the ¹H-NMR data (Fig. 2), proton number 2 appears as a doublet of doublets (dd) at 5.68 ppm with coupling constants J = 13 and 2.7 Hz. The axial and equatorial protons at position 3 are observed at 3.0 and 2.79 ppm, respectively, with corresponding coupling constants of J = 13 and 17 Hz for the axial proton, and J = 2.7 and 17 Hz for the equatorial proton. These values confirm the presence of the flavanone scaffold with an alpha-positioned B-ring. Three methyl signals were appeared at 1.5, 1.58, and 1.67 ppm, corresponding to the C-9, 10, and 4, respectively. The hydroxyl proton at position 5 (H-5) showed a sharp singlet at 12.21 ppm. Proton number 6 (H-6) appears as a singlet at 6.05 ppm. The methylene protons at position 5 appear as two doublets at 4.56 and 4.61 ppm. Aromatic protons on the B-ring are observed at 6.46 (5'), 6.5 (3'), and 7.4 ppm (6'). A detailed summary of the ¹H-NMR assignments is provided in Table 1. Table 1. ¹H-NMR Data for Sophoraflavanone G Position ¹H-NMR (ppm, J in Hz) 2 5.67 (dd 13.2, 2.7) 3 a 3.00 (dd 13.2, 17), 3 b 2.79 (dd 2.7, 17) 4 – 5 12.21 6 6.05 (s) 3ʹ 6.50 (d 2.2) 5ʹ 6.46 (dd 8.3, 2.2) 6ʹ 7.40 (dd 8.2) 1ʺ a 2.64 (dd 1.2) 1ʺ b 2.66 (d 2.3) 2ʺ 2.58 (m) 4ʺ 1.67 (s) 5ʺ 4.56, 4.61 (brs) 6ʺ 2.05 (m) 7ʺ 5.01 (brt) 9ʺ 1.50 (s) 10ʺ 1.58 (s) Antibacterial Activity of Sophoraflavanone G MIC and MBC of Sophoraflavanone G against the selected bacterial strains are shown in Table 2. The compound exhibited potent activity against the all tested Gram-positive bacteria, including MRSA. In contrast, the Gram-negative bacterium, P. aeruginosa PAO1, was unaffected at concentrations up to 1000 µg/mL. Table 2. MIC and MBC of sophoraflavanone G against the tested microorganisms (all values are expressed in µg/ml). Microorganism MIC (µg/mL) MBC (µg/mL) Pseudomonas aeruginosa PAO1 >1000 >1000 Staphylococcus epidermidis DSMZ 3270 7.8 7.8 Listeria monocytogenes 1298 0.98 7.8 Bacillus subtilis 1247 7.8 7.8 Staphylococcus aureus 1431 (MSSA) 7.8 7.8 Staphylococcus aureus 1112 (MRSA) 7.8 7.8 Micrococcus luteus 1110 7.8 7.8 Among Gram-positive bacteria, L. monocytogenes exhibited the greatest sensitivity (MIC = 0.98 µg/mL). Identical MIC and MBC values (7.8 µg/mL) were observed for S. epidermidis , MRSA, MSSA, B. subtilis , and M. luteus . The MBC values confirmed that Sophoraflavanone G has bactericidal activity against all tested Gram-positive bacteria. This lower effect against Gram-negative bacteria could be due to the presence of a rich hydrophilic lipopolysaccharides (LPS) barrier in outer membrane of such pathogens, which prohibits the penetration of strong hydrophobic compounds [33, 34]. Biofilm Inhibition and Synergistic Activity in P. aeruginosa The anti-biofilm potential of Sophoraflavanone G against P. aeruginosa PAO1 was assessed using the crystal violet staining method. As shown in Fig. 3, the compound was ineffective in inhibiting biofilm formation at any tested concentration . In additional experiments, the ability of Sophoraflavanone G to enhance the penetration of tobramycin into mature P. aeruginosa biofilms was evaluated. Co-treatment with tobramycin and 0.5 mg/mL of Sophoraflavanone G showed no significant improvement in biofilm penetration compared to tobramycin alone or untreated controls (Fig. 4). However, at 1 mg/mL, Sophoraflavanone G significantly enhanced the activity of tobramycin, leading to improved antibiotic penetration and increased killing of biofilm-embedded bacteria (P < 0.001). These findings suggest a concentration-dependent synergistic effect between Sophoraflavanone G and tobramycin. In analysis of the biofilm degradation, the intensity of the color formed was so high and there were no differences between the control and treated wells (data are not shown). Hence, the mechanism of this effect is not due to the destruction and removal of the biofilm layer. Inhibitory Effects on Biofilm Formation, Disruption of Constructed Biofilm, and Eliminating Bacteria Protected within the Biofilm of S. epidermidis The results of the compound’s effect on inhibiting S. epidermidis biofilm formation are presented in Fig. 5. Based on Fig. 5, unlike P. aeruginosa , sophoraflavanone G was able to inhibit biofilm formation of S. epidermidis DSMZ 3270, significantly. Interestingly, this effect was also seen on the constructed biofilm of S. epidermidis (Fig. 6). In other words, Sophoraflavanone G at both concentrations of 1/2 MIC and MIC was significantly effective in disrupting the biofilm of S. epidermidis DSMZ 3270. Therefore, it was expected that this compound, even on its own, would be able to destroy bacteria sheltering in biofilms. The results of the compound’s effect on penetrating the biofilm of S. epidermidis are presented in Fig. 7. Based on this figure, sophoraflavanone G , at both MIC and 1/2 MIC concentrations was significantly effective in eliminating bacteria protected within the biofilm. This figure shows that combination treatment with an appropriate antibiotic can lead to the complete elimination of the cells protected within the biofilm. Jia et al. demonstrated that alkaloids from Sophora alopecuroides interfere with AI-2 signaling in S. epidermidis biofilms, suggesting that similar bioactive compounds, such as sophoraflavanone G, may exert anti-biofilm effects by disrupting quorum-sensing pathways [35]. According to the study by Chan et al., Sophoraflavanone G exhibited notable antimicrobial activity against Gram-positive bacteria. However, it demonstrated no antimicrobial effect against Gram-negative bacteria such as Escherichia coli , E. coli O157, Vibrio vulnificus , Shigella , and Salmonella typhi [36]. Therefore, its lack of activity against the resistant P. aeruginosa PAO1 strain observed in this study is understandable. On the other hand, regardless of the Staphylococcal strain tested (MRSA or MSSA), similar results were obtained for MIC and MBC. Tsuchiya et al. confirmed that among the flavanones extracted from Sophora species, Sophoraflavanone G and Exiguaflavanone D exhibited the highest activity against various MRSA strains, with MIC values ranging from 13.3 to 25.6 μg/ml. The MICs of these flavanones against MSSA and MRSA were determined to be similar, suggesting that the anti-MRSA activity of the flavanones is independent of the degree of antibiotic resistance in Staphylococcal strains [37]. However, according to the studies, the MIC values for various S. aureus strains in different studies ranged from 0.05 to 8 μg/ml, which is consistent with the 7.8 μg/ml value obtained in this study [36, 38-40]. In the experiments conducted in this study, the MIC of Sophoraflavanone G against S. epidermidis was found to be 7.8 μg/ml, which closely aligns with the MIC reported by Wan et al. [41]. An et al. also demonstrated the antibacterial activity of prenylated flavonoids from Sophora flavescens against the plant pathogen Xanthomonas oryzae , suggesting a broad-spectrum effect that extends beyond human pathogens [42]. Therefore, the MIC values obtained for Sophoraflavanone G against the microorganisms listed in Table 1 are consistent with the experimental data from previous studies Various studies have proposed antimicrobial mechanisms for the compound Sophoraflavanone G. Tsuchiya and Iinuma investigated the antibacterial activity mechanism of Sophoraflavanone G and suggested that the compound reduces the fluidity of both the outer and inner layers of bacterial membranes. Specifically, the presence of the aliphatic lavandulyl group at the C8 position enables Sophoraflavanone G to interact with bacterial cell membranes more effectively than Naringenin (5,7,4'-trihydroxyflavanone), thereby enhancing its membrane-disruptive activity. Sophoraflavanone G affects membrane fluidity at concentrations ranging from 0.05 to 5 μg/ml, whereas Naringenin requires higher concentrations above 2.5 μg/ml to exhibit similar effects. Notably, Sophoraflavanone G reduces the fluidity of both the outer and inner membrane layers equally, while Naringenin predominantly affects the outer membrane layer more than the inner one [43]. In one study aimed at confirming cell wall disruption in Enterococcus faecium caused by Sophoraflavanone G, the compound was added to bacterial strains at concentrations ranging from 0 to 50 μg/ml. Subsequently, UV absorbance of the supernatant was measured at 260 nm and 280 nm using a spectrophotometer. A significant increase in absorbance was observed following the addition of Sophoraflavanone G, indicating leakage of intracellular components such as proteins and nucleic acids. Based on transmission electron microscopy (TEM) images and additional assays, the proposed antimicrobial mechanism of Sophoraflavanone G in this article involves its binding to bacterial cell wall peptidoglycans, leading to cell wall disintegration and ultimately bacterial cell lysis [26]. These mechanisms were also confirmed by Weng et al. Their comprehensive mechanistic studies revealed that Sophoraflavanone G could destruct MRSA bacterial membrane integrity, increase permeability, and change the membrane potential. It could also disrupt cell wall synthesis, induce hydrolysis in the cell wall, and inhibit construction of bacterial biofilms by reducing the biosynthesis of PIA (the major component in the biofilm matrix of Gram-positive bacteria). In addition, it can interfere with the energy production of MRSA and interrupt the normal physiological activities. In vivo studies have shown that treatment of skin infections caused by MRSA with Sophoraflavanone G, promoted wound healing, and inhibited purulent secretion through remarkable reduction of the bacteria in the infected areas and suppression of the of pro-inflammatory cytokine, IL-6, levels [27]The majority of these antibacterial mechanisms of Sophoraflavanone G were also established by An et al. in the case of Xanthomonas oryzae pv oryzae ( Xoo ) including inhibition of biofilm formation and metabolic activity, reducing extracellular enzyme activity and surface hydrophobicity, inducing the production of ROS, decreasing ATP generation, disrupting membrane potential of mitochondria, and finally triggering apoptosis [42]. Moreover, several studies have investigated the synergistic effect of Sophoraflavanone G in combination with antibiotics. According to the study by Fakhimi et al., the MIC values of Sophoraflavanone G and gentamicin against S. aureus were determined to be 0.05 μg/ml and 32 μg/ml, respectively. However, in the presence of a sub-MIC concentration of Sophoraflavanone G (0.03 μg/ml), the MIC of gentamicin for this strain was reduced from 32 μg/ml to 8 μg/ml—a fourfold decrease. These findings suggest that the antibacterial effect of gentamicin is enhanced by Sophoraflavanone G [38]. Additionally, Sophoraflavanone G and gentamicin demonstrated significant PAE (post-antibiotic effect) and PA-SME (post-antibiotic sub-MIC effect) at the tested concentrations against S. aureus . The prolongation of PAE induced by Sophoraflavanone G and gentamicin was dose-dependent. Moreover, Sophoraflavanone G at sub-MIC concentrations enhanced both the PAE and PA-SME of gentamicin in a dose-dependent manner. The most pronounced potentiating effect for gentamicin was observed at synergistic MIC (8 μg/ml) and half-synergistic MIC (4 μg/ml) levels in the presence of 0.03 μg/ml Sophoraflavanone G (a concentration below its MIC of 0.05 μg/ml). This synergy increased the PA-SME of gentamicin at 4 μg/ml from 15 minutes to 80 minutes—a sixfold extension. Sophoraflavanone G at 0.03 μg/ml not only enhanced gentamicin’s antibacterial activity but also significantly prolonged its PA-SME duration at synergistic MIC levels. At present, the underlying reason for these enhancements is not fully understood. It has been hypothesized that Sophoraflavanone G’s antibacterial effect may be attributed to a reduction in bacterial cell membrane fluidity [44]. Sophoraflavanone G, isolated from Sophora alopecuroides , exhibited a significant synergistic effect with the antibiotic norfloxacin at a low concentration (1 mg/ml) against the fluoroquinolone-resistant S. aureus strain SA1199B (MRSA) under in vitro conditions. The MIC of norfloxacin combined with Sophoraflavanone G was found to be sixteen times lower than that of norfloxacin alone, decreasing from 32 μg/ml to 2 μg/ml. Since the SA1199B strain overexpresses the NorA efflux protein, it is hypothesized that the synergistic mechanism between Sophoraflavanone G and norfloxacin involves inhibition of this pump. Results from ethidium bromide (EtBr) efflux assays supported this hypothesis, showing that Sophoraflavanone G exhibited NorA-efflux inhibitory activity in S. aureus SA1199B compared to the positive control [45]. Moreover, it has been shown that this compound enhances the effect of further antibiotics as either additivity or synergy including ciprofloxacin, erythromycin, fusidic acid, ampicillin and oxacillin [46]. In the experiments conducted in this study, Sophoraflavanone G also exhibited promising synergistic activity with tobramycin against P. aeruginosa PAO1. In a study by Wan et al. Sophoraflavanone G demonstrated notable antibacterial and anti-biofilm activity against S. epidermidis (ATCC 35984, BF⁺). The compound’s anti-biofilm effects against this bacterium were also evaluated at concentrations of 3.125, 6.25, 12.5, 50, and 100 μg/ml. the results revealed that Sophoraflavanone G effectively inhibited biofilm formation of S. epidermidis at all tested concentrations except 3.125 μg/ml [41]. Additionally, Fan et al. showed that alkaloids from Sophora flavescens are effective against metronidazole-resistant Gardnerella vaginalis under both planktonic and biofilm conditions, reinforcing the biofilm-targeting potential of this genus [47]. Future investigations should also explore its mechanism of synergy at the molecular level and evaluate its in vivo therapeutic potential, In this context, Sychrová et al. emphasized the value of prenylated flavonoids like sophoraflavanone G in treating topical infections and promoting wound healing, due to their dual antimicrobial and anti-inflammatory properties [46]. Conclusion In conclusion, sophoraflavanone G is a potent antibacterial agent, especially for Gram-positive bacteria, and a promising anti-biofilm compound, particularly against S. epidermidis . The ability of this compound to penetrate biofilms and kill microorganisms is also a notable point that could provide combination therapy with antibiotics. On the other hand, it has been shown sophoraflavanone G has no cytotoxic effects (up to 8 µg/ml), that is important for wound healing [48]. However, its limited efficacy against Gram-negative bacteria, principally in biofilm-associated states, highlights the need for further structural optimization or smart formulation strategies to enhance its activity. Future investigations should explore its mechanism of synergy and probable drug interactions at the molecular level, possible toxicity to various human cells, and also evaluate its in vivo therapeutic potential. Declarations Declaration of competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding: This work was supported by a grant from Mashhad University of Medical Sciences, Research Council, Mashhad, Iran. The results presented in this paper were part of a student thesis, and the Grant number is 981034. Author Contribution N.Z. Initiated the study, conducted the experimental workG.H. Drafted the original manuscriptA. S .Revised the manuscriptB. F. B. Provided supervisionM.I. Provided supervision, developed the conceptual framework, and performed critical revisions V. S. Developed the overall research objectives and methodologies, provided supervision, and conducted critical revisions Data Availability Data is provided within the manuscript References Abbas A, Barkhouse A, Hackenberger D, Wright GD. Antibiotic resistance: A key microbial survival mechanism that threatens public health. Cell Host Microbe. 2024;32(6):837–51. Bhaskar P, Sahu B. Antimicrobial resistance: global concern and the critical need for new antibiotics. Appl Biology Chem J. 2023;4(1):1–3. Bello OO, et al. Occurrence and Role of Bacterial Biofilms in Different Systems. Acta Microbiol Bulg. 2023;39:239–48. Wang X, Liu M, Yu C, Li J, Zhou X. Biofilm formation: mechanistic insights and therapeutic targets. Mol Biomed. 2023;4(1):49. Shree P, Singh CK, Sodhi KK, Surya JN, Singh DK. Biofilms: Understanding the structure and contribution towards bacterial resistance in antibiotics. Med Microecology. 2023;16:100084. Pokharel K, Dawadi BR, Shrestha LB. Role of biofilm in bacterial infection and antimicrobial resistance. JNMA: J Nepal Med Association. 2022;60(253):836. Ramírez-Larrota JS, Eckhard U. An introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion, Biomolecules , vol. 12, no. 2, p. 306, 2022. Yang J, Xu J-F, Liang S. Antibiotic resistance in Pseudomonas aeruginosa : mechanisms and emerging treatment. Crit Rev Microbiol, pp. 1–19, 2024. Hulen C, Racine P-j, Chevalier S, Feuilloley M, Lomri N-E. Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in Pseudomonas aeruginosa PAO1, Antibiotics , vol. 9, no. 9, p. 596, 2020. Elfadadny A, et al. Antimicrobial resistance of Pseudomonas aeruginosa : navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front Microbiol. 2024;15:1374466. Chabi R, Momtaz H. Virulence factors and antibiotic resistance properties of the Staphylococcus epidermidis strains isolated from hospital infections in Ahvaz, Iran. Trop Med health. 2019;47:1–9. Najar-Peerayeh S, Moghadas AJ, Behmanesh M. Antibiotic susceptibility and mecA frequency in Staphylococcus epidermidis , isolated from intensive care unit patients. Jundishapur J Microbiol. 2014;7(8):e11188. Oliveira F, Melo LD, Cerca N. Relationship between biofilm formation and antibiotic resistance in commensal isolates of Staphylococcus epidermidis , 2013. Lee D-H, Lee K, Kim Y-S, Cha C-J. Comprehensive genomic landscape of antibiotic resistance in Staphylococcus epidermidis , Msystems , vol. 9, no. 6, pp. e00226-24, 2024. Asante J, Hetsa BA, Amoako DG, Abia AL, Bester LA, Essack SY. Genomic analysis of antibiotic-resistant Staphylococcus epidermidis isolates from clinical sources in the Kwazulu-Natal Province, South Africa. Front Microbiol. 2021;12:656306. West KH, et al. Non-native peptides capable of pan-activating the agr quorum sensing system across multiple specificity groups of Staphylococcus epidermidis . ACS Chem Biol. 2021;16(6):1070–8. Eisenbraun EL, Vulpis TD, Prosser BN, Horswill AR, Blackwell HE. Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities. J Am Chem Soc. 2024;146(23):15941–54. Durham PG et al. Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant S taphylococcus aureus biofilms, Biofilm , vol. 3, p. 100049, 2021. Pai L, Patil S, Liu S, Wen F. A growing battlefield in the war against biofilm-induced antimicrobial resistance: insights from reviews on antibiotic resistance. Front Cell Infect Microbiol. 2023;13:1327069. Jo D-M et al. Green Medicine: Advancing Antimicrobial Solutions with Diverse Terrestrial and Marine Plant-Derived Compounds, Processes , vol. 12, no. 11, p. 2316, 2024. Košćak L, Lamovšek J, Đermić E, Prgomet I, Godena S. Microbial and plant-based compounds as alternatives for the control of phytopathogenic bacteria, Horticulturae , vol. 9, no. 10, p. 1124, 2023. Li S, et al. Natural antimicrobials from plants: Recent advances and future prospects. Food Chem. 2024;432:137231. Nwafor IR et al. Plant-derived Bioactive Compounds and Their Mechanistic Roles in Combating Microbial Biofilms. Boozari M, Soltani S, Iranshahi M. Biologically active prenylated flavonoids from the genus Sophora and their structure–activity relationship—A review. Phytother Res. 2019;33(3):546–60. Emami SA, Amin-Ar-Ramimeh E, Ahi A, Bolourian Kashy MR, Schneider B, Iranshahi M. Prenylated flavonoids and flavonostilbenes from Sophora pachycarpa. roots. Pharm Biol. 2007;45(6):453–7. Kim D, Kim K-y. Antibacterial effect of sophoraflavanone G by destroying the cell wall of Enterococcus faecium . J Appl Pharm Sci. 2020;10(9):059–64. Weng Z, et al. Antimicrobial activities of lavandulylated flavonoids in Sophora flavences against methicillin-resistant Staphylococcus aureus via membrane disruption. J Adv Res. 2024;57:197–212. Yuan G et al. One Earth-One Health (OE-OH): Antibacterial Effects of Plant Flavonoids in Combination with Clinical Antibiotics with Various Mechanisms, Antibiotics , vol. 14, no. 1, p. 8, 2024. Fakhimi A, Iranshahi M, Emami SA, Amin-Ar-Ramimeh E, Zarrini G, Shahverdi AR. Sophoraflavanone G from Sophora pachycarpa enhanced the antibacterial activity of gentamycin against Staphylococcus aureus . Z für Naturforschung C. 2006;61:9–10. Cha J-D, Jeong M-R, Jeong S-I, Lee K-Y. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens . J Microbiol Biotechnol. 2007;17(5):858–64. Hwang S-M et al. Antibacterial activity of Sophoraflavanone G isolated from the roots of Sophora flavescens and Red Ginseng Extract, 한국미생물학회 학술대회논문집, pp. 236–236, 2013. Gao Y et al. Sophoraflavanone G: A review of the phytochemistry and pharmacology, Fitoterapia , vol. 177, p. 106080, 2024. Shakeri A, Khakdan F, Soheili V, Sahebkar A, Rassam G, Asili J. Chemical composition, antibacterial activity, and cytotoxicity of essential oil from Nepeta ucrainica L. spp. kopetdaghensis . Ind Crops Prod. 2014;58:315–21. Bashi DS, Dowom SA, Bazzaz BSF, Khanzadeh F, Soheili V, Mohammadpour A. Evaluation, prediction and optimization the ultrasound-assisted extraction method using response surface methodology: antioxidant and biological properties of Stachys parviflora L. Iran J basic Med Sci. 2016;19(5):529. Jia F, Zhou Q, Li X, Zhou X. Total alkaloids of Sophora alopecuroides and matrine inhibit auto-inducer 2 in the biofilms of Staphylococcus epidermidis . Microb Pathog. 2019;136:103698. Chan BC-L, et al. Quick identification of kuraridin, a noncytotoxic anti-MRSA (methicillin-resistant Staphylococcus aureus ) agent from Sophora flavescens using high-speed counter-current chromatography. J Chromatogr B. 2012;880:157–62. Tsuchiya H, et al. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus . J Ethnopharmacol. 1996;50(1):27–34. Fakhimi A, Iranshahi M, Emami SA, Amin-Ar-Ramimeh E, Zarrini G, Shahverdi AR. Sophoraflavanone G from Sophora pachycarpa enhanced the antibacterial activity of gentamycin against Staphylococcus aureus . Z für Naturforschung C. 2006;61:11–2. Oh I, Yang W-Y, Chung S-C, Kim T-Y, Oh K-B, Shin J. In vitro sortase A inhibitory and antimicrobial activity of flavonoids isolated from the roots of Sophora flavescens . Arch Pharm Res. 2011;34:217–22. Yuan G, Guan Y, Yi H, Lai S, Sun Y, Cao S. Antibacterial activity and mechanism of plant flavonoids to gram-positive bacteria predicted from their lipophilicities. Sci Rep. 2021;11(1):10471. Wan C-X, Luo J-G, Ren X-P, Kong L-Y. Interconverting flavonostilbenes with antibacterial activity from Sophora alopecuroides , Phytochemistry , vol. 116, pp. 290–297, 2015. An J-X et al. Antibacterial Activities of Prenylated Flavonoids from Sophora flavences against Xanthomonas oryzae pv oryzae. J Agric Food Chem, 2025. Tsuchiya H, Iinuma M. Reduction of membrane fluidity by antibacterial sophoraflavanone G isolated from Sophora exigua , Phytomedicine , vol. 7, no. 2, pp. 161–165, 2000. Mirjani R, Rafii F, Sharifzadeh M, Amanlou M, Shahverdi AR. Post-antibiotic and post-antibiotic sub-MIC effects of gentamicin, sophoraflavanone G, and their combination against a clinical isolate of Staphylococcus aureus . Asian Biomed. 2010;4:821–6. Sun Z-L, Sun S-C, He J-M, Lan J-E, Gibbons S, Mu Q. Synergism of sophoraflavanone G with norfloxacin against effluxing antibiotic-resistant Staphylococcus aureus . Int J Antimicrob Agents. 2020;56(3):106098. Sychrová A, Škovranová G, Čulenová M, Bittner S, Fialová. Prenylated flavonoids in topical infections and wound healing, Molecules , vol. 27, no. 14, p. 4491, 2022. Fan L, Liu Z, Zhang Z, Bai H. Antimicrobial Effects of Sophora flavescens Alkaloid s on Metronidazole-Resistant Gardnerella vaginalis in Planktonic and Biofilm Conditions. Curr Microbiol. 2023;80(8):263. Kim CS et al. Antimicrobial effect of sophoraflavanone G isolated from Sophora flavescens against mutans streptococci, Anaerobe , vol. 19, pp. 17–21, 2013. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers invited by journal 11 Nov, 2025 Editor invited by journal 22 Sep, 2025 Editor assigned by journal 22 Sep, 2025 Submission checks completed at journal 04 Sep, 2025 First submitted to journal 04 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":12571,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of Sophoraflavanone G\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/aabd7f8c41d0d404b8b387d6.png"},{"id":96746440,"identity":"612fb081-3dfd-4738-bbe3-ae913ba3f1e7","added_by":"auto","created_at":"2025-11-25 16:07:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":221649,"visible":true,"origin":"","legend":"\u003cp\u003eThe ¹H-NMR data of Sophoraflavanone G\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/9dd0910f295fe83ce74b1302.png"},{"id":96914404,"identity":"a4edec7b-9227-45fa-8fa8-81da7bc53c3f","added_by":"auto","created_at":"2025-11-27 14:05:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":151695,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Sophoraflavanone G on the inhibition of biofilm formation in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/a61b08cde332146b051f3a66.png"},{"id":96914108,"identity":"e55173e5-b026-4f8b-8a82-9cd98ecd6546","added_by":"auto","created_at":"2025-11-27 14:05:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":282968,"visible":true,"origin":"","legend":"\u003cp\u003eThe synergistic effect of Tobramycin and sophoraflavanone G (at 0.5 mg/ml and 1 mg/ml concentration) on biofilm penetration of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/f9e390648c6be696b7285d0a.png"},{"id":96914401,"identity":"809f0e2a-25df-4c53-bbb8-af440541b311","added_by":"auto","created_at":"2025-11-27 14:05:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":373506,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of sophoraflavanone G on inhibiting biofilm formation of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e DSMZ 3270\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/de5302403c76411d68e54899.png"},{"id":96746451,"identity":"93fd0168-9975-4567-9a1d-7255d126a909","added_by":"auto","created_at":"2025-11-25 16:07:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":399031,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Sophoraflavanone G on the disruption of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e DSMZ 3270 biofilm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/ed70698ede4ef5433fe6e763.png"},{"id":96914172,"identity":"b81f2501-82c4-42c2-bef2-146e7e4d73e0","added_by":"auto","created_at":"2025-11-27 14:05:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":436585,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Sophoraflavanone G on penetration of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003eDSMZ 3270 biofilm\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/055539ff278b654fd03e18a7.png"},{"id":96922388,"identity":"f243a838-c594-4e6b-b6d0-8091d83eeff2","added_by":"auto","created_at":"2025-11-27 14:19:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2163792,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7299693/v1/734fa03c-deca-4973-8028-0ab292a22527.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antibacterial Evaluation of Sophoraflavanone G from Sophora pachycarpa Against Pseudomonas aeruginosa and Staphylococcus epidermidis Biofilm","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotic resistance (AMR) is a critical global public health issue that threatens the effectiveness of antibiotics in treating bacterial infections. This phenomenon is not only a modern challenge but has ancient origins, as bacteria have evolved resistance mechanisms over millions of years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The widespread use of antibiotics in human medicine, veterinary practice, and agriculture has accelerated the development and spread of resistant bacteria, creating a complex web of resistance elements that are genetically diverse and mechanistically sophisticated [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Biofilm-forming bacteria play a significant role in persistent infections due to their unique structural and functional characteristics that enhance survival against antimicrobial treatments and host defenses. These bacteria, such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, form complex communities encased in an extracellular polymeric substance (EPS) matrix, which not only protects them from antibiotics but also facilitates genetic exchange, including antibiotic resistance genes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, the EPS matrix acts as a physical barrier, creating gradients of nutrients and oxygen that contribute to the metabolic dormancy of some cells, making them less susceptible to antimicrobial agents [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, biofilms can impair the activation of immune responses, such as phagocytosis and the complement system, allowing bacteria to evade host defenses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 is a significant pathogen in clinical settings due to its robust antibiotic resistance mechanisms and ability to cause persistent infections. This bacterium is particularly problematic in hospital environments, where it contributes to nosocomial infections, especially in immunocompromised and cystic fibrosis patients. The resistance of \u003cem\u003eP. aeruginosa\u003c/em\u003e to antibiotics is multifaceted, involving intrinsic mechanisms such as efflux pumps and acquired resistance through gene mutations and adaptive responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, innovative treatment strategies are being explored, including non-antibiotic approaches like quorum sensing inhibition, phage therapy, and nanoparticle-based treatments, although these approaches face challenges related to cost and safety [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eQuorum sensing (QS) in P. aeruginosa plays a critical role in regulating virulence and biofilm formation, significantly contributing to its pathogenicity. This process involves the production of signaling molecules that coordinate gene expression related to virulence factors and biofilm development, enabling the bacteria to adapt and thrive in hostile environments, such as during infections in immunocompromised patients [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Disrupting QS can effectively reduce the pathogenicity of P. aeruginosa without exerting additional selective pressure for antibiotic resistance, as QS inhibitors (QSIs) can attenuate virulence without inhibiting bacterial growth [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, a common skin commensal, has emerged as a significant opportunistic pathogen, particularly in healthcare settings, due to its ability to cause infections such as septicaemia and endocarditis. The prevalence of antibiotic resistance among \u003cem\u003eS. epidermidis\u003c/em\u003e strains is alarming, with studies indicating high resistance rates against penicillin (95.65%), tetracycline (91.30%), and methicillin (92.2%) among hospital isolates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The bacterium's capacity to form biofilms enhances its virulence and resistance to antibiotic treatment, complicating management strategies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, the presence of resistance genes, such as mecA and various efflux pumps, contributes to its multi-drug resistant phenotype, making infections difficult to treat [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This multidrug resistance poses significant challenges for treatment, particularly in immunocompromised patients, highlighting the need for ongoing surveillance and novel therapeutic strategies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eS. epidermidis\u003c/em\u003e, QS is a critical mechanism that regulates biofilm formation and virulence, primarily through the accessory gene regulator (AGR) system. This system is activated by autoinducing peptides (AIPs) that bind to the AgrC receptor, leading to the expression of virulence factors and biofilm dispersal agents such as phenol-soluble modulins [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Inhibiting QS in \u003cem\u003eS. epidermidis\u003c/em\u003e is a promising strategy to combat biofilm-associated infections, particularly those involving drug-resistant strains. Various approaches have been explored, including the use of synthetic peptides and natural compounds [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConventional antibiotics are often ineffective against biofilms due to several inherent challenges. Biofilms, which are communities of bacteria encased in a protective matrix, exhibit reduced susceptibility to antibiotics as these drugs typically target planctonic cells, while many cells within biofilms are metabolically inactive or in a persister state. In addition, the resistance is amplified by efflux pumps and physiological changes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe exploration of plant-derived compounds as alternative antimicrobial agents has gained significant traction due to the rising threat of AMR and the limitations of conventional antibiotics. Various studies highlight the diverse antimicrobial properties of these compounds, which include essential oils, extracts, and bioactive substances from both medicinal and non-medicinal plants, as well as marine sources [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These plant-derived agents exhibit multi-target mechanisms, making them less prone to resistance compared to traditional single-target antibiotics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, they have shown efficacy against biofilms, which are notoriously difficult to treat due to their protective matrices [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eSophora pachycarpa\u003c/em\u003e, a member of the Fabaceae family, has been utilized in traditional medicine for its diverse therapeutic properties, primarily attributed to its rich content of flavonoids, particularly prenylated flavonoids. Research has identified several bioactive compounds, including Sophoraflavanone G and Sophoraisoflavanone A, which exhibit significant biological activities such as anti-inflammatory, antibacterial, and cytotoxic effects [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Sophoraflavanone G, a prenylated flavonoid predominantly isolated from plants of the genus \u003cem\u003eSophora\u003c/em\u003e, has garnered attention for its notable antimicrobial properties. Recent studies have demonstrated its efficacy against a range of bacterial pathogens, including methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and \u003cem\u003eEnterococcus faecium\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Its mode of action is believed to involve disruption of bacterial cell membrane integrity and interference with peptidoglycan synthesis, making it a promising candidate for novel antimicrobial therapies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite growing evidence supporting the broad-spectrum antibacterial activity of Sophoraflavanone G, its specific effects on biofilm formation and eradication remain inadequately characterizedand the potential of Sophoraflavanone G to act synergistically with conventional antibiotics, particularly tobramycin against \u003cem\u003eP. aeruginosa\u003c/em\u003e has not been systematically evaluated. These knowledge gaps underscore the necessity for targeted investigations into the anti-biofilm and combinatory antibacterial properties of this compound, especially in the context of biofilm-mediated antimicrobial resistance. This study, tries to introduce the ability of Sophoraflavanone G in inhibition and disruption of biofilm, in both Gram-positive and Gram-negative bacteria, including \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e, as well as its capacity to potentiate the effect of antibiotics.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals and Materials\u003c/h2\u003e\u003cp\u003eAll solvents and reagents used in this study were of analytical grade. Silica gel GF254 plates and silica gel (230\u0026ndash;400 mesh) for column chromatography were obtained from Merck (Germany). Ethyl acetate, petroleum ether (60\u0026ndash;80\u0026deg;C), dichloromethane, methanol, and acetone were purchased from Dr. Mojallali Co. (Iran). Vanillin, sulfuric acid, sodium chloride (NaCl), glucose, crystal violet, and 2,3,5-triphenyl tetrazolium chloride (TTC) were also sourced from Merck (Germany). Dimethyl sulfoxide (DMSO) was obtained from Merck (Germany), and absolute ethanol was purchased from Simin Tak (Iran). Tryptic Soy Agar (TSA) was acquired from Himedia (India), while Mueller-Hinton Broth (MHB) was provided by Liofilchem (Italy). Tobramycin was sourced from Sigma (USA), and cloxacillin was obtained from Farabi (Iran).\u003c/p\u003e\u003cp\u003eThe bacterial strains used in this study included \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 (Wild type Nottingham), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (DSMZ 3270, Germany), \u003cem\u003eListeria monocytogenes\u003c/em\u003e (PTCC 1298, Iran), \u003cem\u003eBacillus subtilis\u003c/em\u003e (PTCC 1247, Iran), methicillin-sensitive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MSSA, PTCC 1431, Iran), methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA, PTCC 1112, Iran), and \u003cem\u003eMicrococcus luteus\u003c/em\u003e (PTCC 1110, Iran). All strains were maintained and handled according to standard microbiological practices.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExtraction and Purification\u003c/h3\u003e\n\u003cp\u003eThe roots of \u003cem\u003eS. pachycarpa\u003c/em\u003e were collected in June 2019 from the Kalat region, Razavi Khorasan Province, Iran. Botanical identification was performed by Mrs. Soozani, and a voucher specimen (No. 13275) was deposited in the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. The air-dried roots (1350 g) were powdered using an electric grinder and macerated in 4 L of acetone for 24 hours at room temperature. The extract was filtered and concentrated under reduced pressure using a rotary evaporator to afford a reddish-brown residue (35.5 g).\u003c/p\u003e\n\u003ch3\u003eIsolation and Purification of Sophoraflavanone G and Structure Elucidation\u003c/h3\u003e\n\u003cp\u003eThe crude extract was subjected to column chromatography on silica gel using a polarity-gradient elution system comprising petroleum ether, ethyl acetate, and methanol. A total of 65 fractions (200 mL each) were collected and concentrated under reduced pressure. The solvent ratios used for each fraction are listed in Table\u0026nbsp;5. Further purification of the target compound was achieved by preparative thin-layer chromatography (PTLC). Development was carried out in a petroleum ether:ethyl acetate (6:4) system. The resolved band corresponding to the standard was visualized under UV light, marked, scraped off, and extracted with acetone. The extract was filtered and the solvent was removed under reduced pressure to obtain the purified compound. The structure of compound was confirmed through spectral interpretation (1H-NMR) and comparison with previously published data [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of MIC and MBC\u003c/h3\u003e\n\u003cp\u003eThe MIC (Minimum Inhibitory Concentration) of sophoraflavanone G was tested against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eL. monocytogenes\u003c/em\u003e, \u003cem\u003eM. luteus\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1. A 2 mg sample of the compound was dissolved in 70 \u0026micro;l DMSO (3.5%) and 930 \u0026micro;l MHB (Mueller-Hinton Broth). Serial dilutions (1000\u0026thinsp;\u0026minus;\u0026thinsp;0.49 \u0026micro;g/ml) were prepared in a 96-well plate, with each concentration tested in duplicate. Positive control wells contained only separate bacteria in MHB; negative controls contained no bacteria. After adding 20 \u0026micro;l bacterial suspension (10⁶ CFU/ml), the plates were incubated for 24 h. Then, 20 \u0026micro;l TTC (5 mg/ml) was added and incubated for another 1 h. The lowest concentration with no color change was reported as MIC. Following MIC assays, samples from wells with no red color were streaked on TSA agar and incubated for 24 h. The lowest concentration with no bacterial growth was reported as MBC (Minimum Bactericidal Concentration).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiofilm Assays Involving\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e \u003cb\u003ePAO1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the inhibitory effect of sophoraflavanone G on biofilm formation by \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1, a suspension containing 10⁶ CFU/mL of the bacterium was prepared. A total of 20 \u0026micro;L of this suspension was added to 180 \u0026micro;L of glucose-enriched Mueller-Hinton broth (MHB) containing sophoraflavanone G at concentrations corresponding to \u0026frac12; MIC (0.5 mg/mL). For the positive control, 180 \u0026micro;L of medium and 20 \u0026micro;L of bacterial suspension were used. The negative control wells contained only the medium. Plates were incubated at 37\u0026deg;C to allow biofilm formation.\u003c/p\u003e\u003cp\u003eAfter 24 and 36 hours, the content of each well was gently aspirated and refreshed with medium containing the same concentration of sophoraflavanone G. At 48 hours, the wells were emptied and washed twice with 200 \u0026micro;L of normal saline. Subsequently, 50 \u0026micro;L of 0.03% crystal violet solution was added to each well and allowed to incubate for 10 minutes. Excess dye was then removed, and wells were washed at least twice to remove unbound stain. After drying, 200 \u0026micro;L of 95% ethanol was added to each well to solubilize the bound dye, and absorbance was measured at 590 nm using an ELISA reader to quantify the biomass of the formed biofilm.\u003c/p\u003e\u003cp\u003eTo investigate whether sophoraflavonone G facilitates the penetration of tobramycin into the constructed biofilm, first, biofilms were allowed to develop by adding 20 \u0026micro;L of bacterial suspension (10⁶ CFU/mL) to 180 \u0026micro;L of glucose-supplemented MHB in 96-well plates, followed by a 24-hour incubation. After this period, the medium was removed and replaced with fresh medium. At 36 hours, the wells were treated with either 1 mg/mL or 0.5 mg/mL of sophoraflavanone G in combination with tobramycin at its MIC level (2 \u0026micro;g/mL). At the 48-hour mark, the wells were washed and then received 180 \u0026micro;L of enriched medium and 20 \u0026micro;L of 5 mg/mL tetrazolium salt (TTC). Following a 4-hour incubation, absorbance at 450 nm was measured to determine metabolic activity, reflecting penetration and antibacterial activity within the biofilm. Wells treated with tobramycin only were used as control (2 \u0026micro;g/mL). The results compared with the positive controls which received no treatment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiofilm Assays Involving\u003c/b\u003e \u003cb\u003eS. epidermidis\u003c/b\u003e \u003cb\u003eDSMZ 3270\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn evaluating the ability of sophoraflavanone G to inhibit biofilm formation by \u003cem\u003eS. epidermidis\u003c/em\u003e, a suspension containing 10⁶ CFU/mL was added (20 \u0026micro;L) to 180 \u0026micro;L of glucose-enriched MHB containing sophoraflavanone G at \u0026frac12; MIC (3.9 \u0026micro;g/mL) and MIC (7.8 \u0026micro;g/mL). The plates were incubated, and after 24, and 36 hours, the medium was gently replaced with fresh medium of identical composition, preserving any forming biofilm. After 48 hours, the wells were washed and stained with crystal violet, followed by measurement of absorbance at 590 nm.\u003c/p\u003e\u003cp\u003eIn evaluating biofilm disruption, 20 \u0026micro;L of the bacterial suspension was combined with 180 \u0026micro;L of glucose-enriched MHB in 96-well plates. After 24-hours incubation, the medium was replaced, and biofilms were allowed to continue forming. At 36 hours, the wells were treated with sophoraflavanone G at \u0026frac12; MIC and MIC concentrations. Positive controls received only fresh medium. After 48 hours, standard crystal violet staining was carried out and absorbance was measured at 590 nm.\u003c/p\u003e\u003cp\u003eFor determining compound penetration into established biofilms, the same procedure was followed, with treatments administered with the similar concentrations of sophoraflavanone G, after 36 hours. Again, at 48 hours, all wells were emptied and received 180 \u0026micro;L of fresh glucose-enriched MHB along with 20 \u0026micro;L of 5 mg/mL TTC solution. After a 4-hour incubation period, absorbance was read at 450 nm to assess bacterial metabolic activity within the biofilm matrix, serving as an indirect indicator of drug penetration.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe results of each experiment were evaluated after three repetitions as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. All data were analyzed by SPSS software (version 27.0.1) using one-way ANOVA (Tamhane/Tukey's Multiple Comparison Tests) at significant level of p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals. All the plant species were collected from various locations by Dr. Milad Iranshahy following proper guidelines and legislation procedures and identified by Ms. Souzani (MSc of Systematic Botany, Department of Pharmacognosy, School of Pharmacy) and have been deposited at the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences, Iran. No specific license was required for the collection of the plants.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eSophoraflavanone G characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSophoraflavanone G, is a bioactive compound that exhibits significant antibacterial properties, particularly against \u003cem\u003eE. faecium\u003c/em\u003e, by disrupting the bacterial cell wall. This disruption is evidenced by the compound\u0026apos;s ability to bind to peptidoglycans, leading to cell wall damage and subsequent cell lysis, as confirmed by transmission electron microscopy [26]. Additionally, Sophoraflavanone G demonstrates synergistic antibacterial effects when combined with other antimicrobial agents, such as ampicillin and gentamicin, against various oral bacteria, significantly reducing the minimum inhibitory and bactericidal concentrations required for efficacy [30, 31]. Despite its promising pharmacological activities, including anti-inflammatory and antiproliferative effects, SFG\u0026apos;s clinical application is limited by its poor water solubility and bioavailability, necessitating further research to enhance its therapeutic potential [32]. In this study, Sophoraflavanone G was isolated and purified using Silica gel column chromatography and preparative thin-layer chromatography (PTLC) using a petroleum ether:ethyl acetate system at a 6:4 ratio. The chemical structure of the compound is shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003eBased on the \u0026sup1;H-NMR data (Fig. 2), proton number 2 appears as a doublet of doublets (dd) at 5.68 ppm with coupling constants \u003cem\u003eJ\u003c/em\u003e = 13 and 2.7 Hz. The axial and equatorial protons at position 3 are observed at 3.0 and 2.79 ppm, respectively, with corresponding coupling constants of \u003cem\u003eJ\u003c/em\u003e = 13 and 17 Hz for the axial proton, and \u003cem\u003eJ\u003c/em\u003e = 2.7 and 17 Hz for the equatorial proton. These values confirm the presence of the flavanone scaffold with an alpha-positioned B-ring.\u003c/p\u003e\n\u003cp\u003eThree methyl signals were appeared at 1.5, 1.58, and 1.67 ppm, corresponding to the C-9, 10, and 4, respectively. The hydroxyl proton at position 5 (H-5) showed a sharp singlet at 12.21 ppm. Proton number 6 (H-6) appears as a singlet at 6.05 ppm. The methylene protons at position 5 appear as two doublets at 4.56 and 4.61 ppm. Aromatic protons on the B-ring are observed at 6.46 (5\u0026apos;), 6.5 (3\u0026apos;), and 7.4 ppm (6\u0026apos;). A detailed summary of the \u0026sup1;H-NMR assignments is provided in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003e\u0026sup1;H-NMR Data for Sophoraflavanone G\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"496\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026sup1;H-NMR (ppm, \u003cem\u003eJ\u003c/em\u003e in Hz)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.67 (dd 13.2, 2.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.00 (dd 13.2, 17),\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.79 (dd 2.7, 17)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.05 (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3ʹ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.50 (d 2.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5ʹ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.46 (dd 8.3, 2.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6ʹ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.40 (dd 8.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1ʺ a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.64 (dd 1.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1ʺ b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.66 (d 2.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.58 (m)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.67 (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.56, 4.61 (brs)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.05 (m)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.01 (brt)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.50 (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10ʺ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.58 (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial Activity of Sophoraflavanone G\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMIC and MBC of Sophoraflavanone G against the selected bacterial strains are shown in Table 2. The compound exhibited potent activity against the all tested Gram-positive bacteria, including MRSA. In contrast, the Gram-negative bacterium, \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1, was unaffected at concentrations up to 1000 \u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eMIC and MBC of sophoraflavanone G against the tested microorganisms (all values are expressed in \u0026micro;g/ml).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"664\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroorganism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMBC (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e DSMZ 3270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eListeria monocytogenes\u003c/em\u003e 1298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e 1247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e 1431 (MSSA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e 1112 (MRSA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eMicrococcus luteus\u003c/em\u003e 1110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAmong Gram-positive bacteria, \u003cem\u003eL. monocytogenes\u003c/em\u003e exhibited the greatest sensitivity (MIC = 0.98 \u0026micro;g/mL). Identical MIC and MBC values (7.8 \u0026micro;g/mL) were observed for\u003cem\u003e\u0026nbsp;S. epidermidis\u003c/em\u003e, MRSA, MSSA, \u003cem\u003eB. subtilis\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;M. luteus\u003c/em\u003e. The MBC values confirmed that Sophoraflavanone G has bactericidal activity against all tested Gram-positive bacteria. This lower effect against Gram-negative bacteria could be due to the presence of a rich hydrophilic lipopolysaccharides (LPS) barrier in outer membrane of such pathogens, which prohibits the penetration of strong hydrophobic compounds [33, 34].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm Inhibition and Synergistic Activity in\u003cem\u003e\u0026nbsp;P. aeruginosa\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anti-biofilm potential of Sophoraflavanone G against \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 was assessed using the crystal violet staining method. As shown in Fig. 3, the compound was ineffective in inhibiting biofilm formation at any tested concentration\u003cins cite=\"mailto:Vahid%20Soheili\" datetime=\"2025-07-20T09:16\"\u003e.\u0026nbsp;\u003c/ins\u003e\u003c/p\u003e\n\u003cp\u003eIn additional experiments, the ability of Sophoraflavanone G to enhance the penetration of tobramycin into mature \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilms was evaluated. Co-treatment with tobramycin and 0.5 mg/mL of Sophoraflavanone G showed no significant improvement in biofilm penetration compared to tobramycin alone or untreated controls (Fig. 4).\u003c/p\u003e\n\u003cp\u003eHowever, at 1 mg/mL, Sophoraflavanone G significantly enhanced the activity of tobramycin, leading to improved antibiotic penetration and increased killing of biofilm-embedded bacteria (P \u0026lt; 0.001). These findings suggest a concentration-dependent synergistic effect between Sophoraflavanone G and tobramycin.\u003cins cite=\"mailto:Vahid%20Soheili\" datetime=\"2025-07-20T09:22\"\u003e\u0026nbsp;\u003c/ins\u003eIn analysis of the biofilm degradation, the intensity of the color formed was so high and there were no differences between the control and treated wells (data are not shown). \u0026nbsp;Hence, the mechanism of this effect is not due to the destruction and removal of the biofilm layer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibitory Effects on Biofilm Formation, Disruption of Constructed Biofilm, and Eliminating Bacteria Protected within the Biofilm of\u003cem\u003e\u0026nbsp;S. epidermidis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the compound\u0026rsquo;s effect on inhibiting \u003cem\u003eS. epidermidis\u003c/em\u003e biofilm formation are presented in Fig. 5. Based on Fig. 5, unlike \u003cem\u003eP. aeruginosa\u003c/em\u003e, sophoraflavanone G was able to inhibit biofilm formation of\u003cem\u003e\u0026nbsp;S. epidermidis\u0026nbsp;\u003c/em\u003eDSMZ 3270, significantly. Interestingly, this effect was also seen on the constructed biofilm of \u003cem\u003eS. epidermidis\u0026nbsp;\u003c/em\u003e(Fig. 6). In other words, Sophoraflavanone G at both concentrations of 1/2 MIC and MIC was significantly effective in disrupting the biofilm of \u003cem\u003eS. epidermidis\u003c/em\u003e DSMZ 3270.\u003c/p\u003e\n\u003cp\u003eTherefore, it was expected that this compound, even on its own, would be able to destroy bacteria sheltering in biofilms. The results of the compound\u0026rsquo;s effect on penetrating the biofilm of \u003cem\u003eS. epidermidis\u003c/em\u003e are presented in Fig. 7.\u003c/p\u003e\n\u003cp\u003eBased on this figure, sophoraflavanone G\u003cins cite=\"mailto:Vahid%20Soheili\" datetime=\"2025-07-20T09:45\"\u003e,\u003c/ins\u003e at both MIC and 1/2 MIC concentrations was significantly effective in eliminating bacteria protected within the biofilm. This figure shows that combination treatment with an appropriate antibiotic can lead to the complete elimination of the cells protected within the biofilm.\u003c/p\u003e\n\u003cp\u003eJia et al. demonstrated that alkaloids from \u003cem\u003eSophora alopecuroides\u003c/em\u003e interfere with AI-2 signaling in \u003cem\u003eS. epidermidis\u0026nbsp;\u003c/em\u003ebiofilms, suggesting that similar bioactive compounds, such as sophoraflavanone G, may exert anti-biofilm effects by disrupting quorum-sensing pathways\u0026nbsp;[35].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to the study by Chan et al., Sophoraflavanone G exhibited notable antimicrobial activity against Gram-positive bacteria. However, it demonstrated no antimicrobial effect against Gram-negative bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e,\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003eO157,\u003cem\u003e\u0026nbsp;Vibrio vulnificus\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Shigella\u003c/em\u003e, and \u003cem\u003eSalmonella typhi\u0026nbsp;\u003c/em\u003e[36]. Therefore, its lack of activity against the resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e PAO1 strain observed in this study is understandable.\u003c/p\u003e\n\u003cp\u003eOn the other hand, regardless of the Staphylococcal strain tested (MRSA or MSSA), similar results were obtained for MIC and MBC. Tsuchiya et al. confirmed that among the flavanones extracted from \u003cem\u003eSophora\u003c/em\u003e species, Sophoraflavanone G and Exiguaflavanone D exhibited the highest activity against various MRSA strains, with MIC values ranging from 13.3 to 25.6 \u0026mu;g/ml. The MICs of these flavanones against MSSA and MRSA were determined to be similar, suggesting that the anti-MRSA activity of the flavanones is independent of the degree of antibiotic resistance in Staphylococcal strains\u0026nbsp;[37]. However, according to the studies, the MIC values for various \u003cem\u003eS. aureus\u003c/em\u003e strains in different studies ranged from 0.05 to 8 \u0026mu;g/ml, which is consistent with the 7.8 \u0026mu;g/ml value obtained in this study\u0026nbsp;[36, 38-40].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the experiments conducted in this study, the MIC of Sophoraflavanone G against \u003cem\u003eS. epidermidis\u003c/em\u003e was found to be 7.8 \u0026mu;g/ml, which closely aligns with the MIC reported by Wan et al.\u0026nbsp;[41]. An et al. also demonstrated the antibacterial activity of prenylated flavonoids from \u003cem\u003eSophora flavescens\u003c/em\u003e against the plant pathogen \u003cem\u003eXanthomonas oryzae\u003c/em\u003e, suggesting a broad-spectrum effect that extends beyond human pathogens\u0026nbsp;[42]. Therefore, the MIC values obtained for Sophoraflavanone G against the microorganisms listed in Table 1 are consistent with the experimental data from previous studies\u003c/p\u003e\n\u003cp\u003eVarious studies have proposed antimicrobial mechanisms for the compound Sophoraflavanone G. Tsuchiya and Iinuma investigated the antibacterial activity mechanism of Sophoraflavanone G and suggested that the compound reduces the fluidity of both the outer and inner layers of bacterial membranes. Specifically, the presence of the aliphatic lavandulyl group at the C8 position enables Sophoraflavanone G to interact with bacterial cell membranes more effectively than Naringenin (5,7,4\u0026apos;-trihydroxyflavanone), thereby enhancing its membrane-disruptive activity. Sophoraflavanone G affects membrane fluidity at concentrations ranging from 0.05 to 5 \u0026mu;g/ml, whereas Naringenin requires higher concentrations above 2.5 \u0026mu;g/ml to exhibit similar effects. Notably, Sophoraflavanone G reduces the fluidity of both the outer and inner membrane layers equally, while Naringenin predominantly affects the outer membrane layer more than the inner one\u0026nbsp;[43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn one study aimed at confirming cell wall disruption in \u003cem\u003eEnterococcus faecium\u003c/em\u003e caused by Sophoraflavanone G, the compound was added to bacterial strains at concentrations ranging from 0 to 50 \u0026mu;g/ml. Subsequently, UV absorbance of the supernatant was measured at 260 nm and 280 nm using a spectrophotometer. A significant increase in absorbance was observed following the addition of Sophoraflavanone G, indicating leakage of intracellular components such as proteins and nucleic acids. Based on transmission electron microscopy (TEM) images and additional assays, the proposed antimicrobial mechanism of Sophoraflavanone G in this article involves its binding to bacterial cell wall peptidoglycans, leading to cell wall disintegration and ultimately bacterial cell lysis\u0026nbsp;[26].\u003c/p\u003e\n\u003cp\u003eThese mechanisms were also confirmed by Weng et al. Their comprehensive\u0026nbsp;mechanistic studies revealed that\u0026nbsp;Sophoraflavanone G\u0026nbsp;could destruct MRSA bacterial membrane integrity, increase permeability, and change the membrane potential. It could also disrupt cell wall synthesis, induce hydrolysis in the cell wall, and inhibit construction of bacterial biofilms\u0026nbsp;by reducing the biosynthesis of PIA (the major component in the biofilm matrix of Gram-positive bacteria). In addition, it can interfere with the energy production of MRSA and interrupt the normal physiological activities. In vivo studies have shown that treatment of skin infections caused by MRSA with\u0026nbsp;Sophoraflavanone G,\u0026nbsp;promoted wound healing, and inhibited purulent secretion through remarkable reduction of the bacteria in the infected areas and suppression of the of pro-inflammatory cytokine, IL-6, levels\u0026nbsp;[27]The majority of these antibacterial mechanisms of\u0026nbsp;Sophoraflavanone G\u0026nbsp;were also established by An et al. in the case of\u0026nbsp;\u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv \u003cem\u003eoryzae\u003c/em\u003e (\u003cem\u003eXoo\u003c/em\u003e) including inhibition of biofilm formation \u0026nbsp;and metabolic activity, reducing extracellular enzyme activity and surface hydrophobicity, inducing the production of ROS, decreasing ATP generation, disrupting membrane potential of mitochondria, and finally triggering apoptosis\u0026nbsp;[42].\u003c/p\u003e\n\u003cp\u003eMoreover, several studies have investigated the synergistic effect of Sophoraflavanone G in combination with antibiotics. According to the study by Fakhimi et al., the MIC values of Sophoraflavanone G and gentamicin against \u003cem\u003eS. aureus\u003c/em\u003e were determined to be 0.05 \u0026mu;g/ml and 32 \u0026mu;g/ml, respectively. However, in the presence of a sub-MIC concentration of Sophoraflavanone G (0.03 \u0026mu;g/ml), the MIC of gentamicin for this strain was reduced from 32 \u0026mu;g/ml to 8 \u0026mu;g/ml\u0026mdash;a fourfold decrease. These findings suggest that the antibacterial effect of gentamicin is enhanced by Sophoraflavanone G\u0026nbsp;[38].\u003c/p\u003e\n\u003cp\u003eAdditionally, Sophoraflavanone G and gentamicin demonstrated significant PAE (post-antibiotic effect) and PA-SME (post-antibiotic sub-MIC effect) at the tested concentrations against \u003cem\u003eS. aureus\u003c/em\u003e. The prolongation of PAE induced by Sophoraflavanone G and gentamicin was dose-dependent. Moreover, Sophoraflavanone G at sub-MIC concentrations enhanced both the PAE and PA-SME of gentamicin in a dose-dependent manner. The most pronounced potentiating effect for gentamicin was observed at synergistic MIC (8 \u0026mu;g/ml) and half-synergistic MIC (4 \u0026mu;g/ml) levels in the presence of 0.03 \u0026mu;g/ml Sophoraflavanone G (a concentration below its MIC of 0.05 \u0026mu;g/ml). This synergy increased the PA-SME of gentamicin at 4 \u0026mu;g/ml from 15 minutes to 80 minutes\u0026mdash;a sixfold extension. Sophoraflavanone G at 0.03 \u0026mu;g/ml not only enhanced gentamicin\u0026rsquo;s antibacterial activity but also significantly prolonged its PA-SME duration at synergistic MIC levels. At present, the underlying reason for these enhancements is not fully understood. It has been hypothesized that Sophoraflavanone G\u0026rsquo;s antibacterial effect may be attributed to a reduction in bacterial cell membrane fluidity\u0026nbsp;[44].\u003c/p\u003e\n\u003cp\u003eSophoraflavanone G, isolated from \u003cem\u003eSophora alopecuroides\u003c/em\u003e, exhibited a significant synergistic effect with the antibiotic norfloxacin at a low concentration (1 mg/ml) against the fluoroquinolone-resistant \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003estrain SA1199B (MRSA) under in vitro conditions. The MIC of norfloxacin combined with Sophoraflavanone G was found to be sixteen times lower than that of norfloxacin alone, decreasing from 32 \u0026mu;g/ml to 2 \u0026mu;g/ml. Since the SA1199B strain overexpresses the NorA efflux protein, it is hypothesized that the synergistic mechanism between Sophoraflavanone G and norfloxacin involves inhibition of this pump. Results from ethidium bromide (EtBr) efflux assays supported this hypothesis, showing that Sophoraflavanone G exhibited NorA-efflux inhibitory activity in \u003cem\u003eS. aureus\u003c/em\u003e SA1199B compared to the positive control\u0026nbsp;[45]. Moreover, it has been shown that this compound enhances the effect of further antibiotics as either additivity or synergy including ciprofloxacin, erythromycin, fusidic acid, ampicillin and oxacillin\u0026nbsp;[46]. In the experiments conducted in this study, Sophoraflavanone G also exhibited promising synergistic activity with tobramycin against \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003ePAO1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn a study by Wan et al. Sophoraflavanone G demonstrated notable antibacterial and anti-biofilm activity against \u003cem\u003eS. epidermidis\u003c/em\u003e (ATCC 35984, BF⁺). The compound\u0026rsquo;s anti-biofilm effects against this bacterium were also evaluated\u003cspan dir=\"RTL\"\u003e\u003cspan\u003e\u003cins cite=\"mailto:Vahid%20Soheili\" datetime=\"2025-07-20T13:01\"\u003e\u0026nbsp;\u003c/ins\u003e\u003c/span\u003e\u003c/span\u003eat concentrations of 3.125, 6.25, 12.5, 50, and 100 \u0026mu;g/ml. the results revealed that Sophoraflavanone G effectively inhibited biofilm formation of \u003cem\u003eS. epidermidis\u003c/em\u003e at all tested concentrations except 3.125 \u0026mu;g/ml [41]. Additionally, Fan et al. showed that alkaloids from \u003cem\u003eSophora flavescens\u003c/em\u003e are effective against metronidazole-resistant \u003cem\u003eGardnerella vaginalis\u003c/em\u003e under both planktonic and biofilm conditions, reinforcing the biofilm-targeting potential of this genus [47]. Future investigations should also explore its mechanism of synergy at the molecular level and evaluate its in vivo therapeutic potential, In this context, Sychrov\u0026aacute; et al. emphasized the value of prenylated flavonoids like sophoraflavanone G in treating topical infections and promoting wound healing, due to their dual antimicrobial and anti-inflammatory properties [46].\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, sophoraflavanone G is a potent antibacterial agent, especially for Gram-positive bacteria, and a promising anti-biofilm compound, particularly against \u003cem\u003eS. epidermidis\u003c/em\u003e. The ability of this compound to penetrate biofilms and kill microorganisms is also a notable point that could provide combination therapy with antibiotics. On the other hand, it has been shown sophoraflavanone G has no cytotoxic effects (up to 8 \u0026micro;g/ml), that is important for wound healing [48]. However, its limited efficacy against Gram-negative bacteria, principally in biofilm-associated states, highlights the need for further structural optimization or smart formulation strategies to enhance its activity. Future investigations should explore its mechanism of synergy and probable drug interactions at the molecular level, possible toxicity to various human cells, and also evaluate its in vivo therapeutic potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by a grant from Mashhad University of Medical Sciences, Research Council, Mashhad, Iran. The results presented in this paper were part of a student thesis, and the Grant number is 981034.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eN.Z. Initiated the study, conducted the experimental workG.H. Drafted the original manuscriptA. S .Revised the manuscriptB. F. B. Provided supervisionM.I. Provided supervision, developed the conceptual framework, and performed critical revisions V. S. Developed the overall research objectives and methodologies, provided supervision, and conducted critical revisions\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas A, Barkhouse A, Hackenberger D, Wright GD. Antibiotic resistance: A key microbial survival mechanism that threatens public health. Cell Host Microbe. 2024;32(6):837\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhaskar P, Sahu B. Antimicrobial resistance: global concern and the critical need for new antibiotics. Appl Biology Chem J. 2023;4(1):1\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBello OO, et al. Occurrence and Role of Bacterial Biofilms in Different Systems. Acta Microbiol Bulg. 2023;39:239\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Liu M, Yu C, Li J, Zhou X. Biofilm formation: mechanistic insights and therapeutic targets. Mol Biomed. 2023;4(1):49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShree P, Singh CK, Sodhi KK, Surya JN, Singh DK. Biofilms: Understanding the structure and contribution towards bacterial resistance in antibiotics. Med Microecology. 2023;16:100084.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePokharel K, Dawadi BR, Shrestha LB. Role of biofilm in bacterial infection and antimicrobial resistance. JNMA: J Nepal Med Association. 2022;60(253):836.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRam\u0026iacute;rez-Larrota JS, Eckhard U. An introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion, \u003cem\u003eBiomolecules\u003c/em\u003e, vol. 12, no. 2, p. 306, 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang J, Xu J-F, Liang S. Antibiotic resistance in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e: mechanisms and emerging treatment. Crit Rev Microbiol, pp. 1\u0026ndash;19, 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHulen C, Racine P-j, Chevalier S, Feuilloley M, Lomri N-E. Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1, \u003cem\u003eAntibiotics\u003c/em\u003e, vol. 9, no. 9, p. 596, 2020.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElfadadny A, et al. Antimicrobial resistance of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e: navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front Microbiol. 2024;15:1374466.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChabi R, Momtaz H. Virulence factors and antibiotic resistance properties of the \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e strains isolated from hospital infections in Ahvaz, Iran. Trop Med health. 2019;47:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNajar-Peerayeh S, Moghadas AJ, Behmanesh M. Antibiotic susceptibility and mecA frequency in \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, isolated from intensive care unit patients. Jundishapur J Microbiol. 2014;7(8):e11188.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOliveira F, Melo LD, Cerca N. Relationship between biofilm formation and antibiotic resistance in commensal isolates of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, 2013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee D-H, Lee K, Kim Y-S, Cha C-J. Comprehensive genomic landscape of antibiotic resistance in \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, \u003cem\u003eMsystems\u003c/em\u003e, vol. 9, no. 6, pp. e00226-24, 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsante J, Hetsa BA, Amoako DG, Abia AL, Bester LA, Essack SY. Genomic analysis of antibiotic-resistant \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e isolates from clinical sources in the Kwazulu-Natal Province, South Africa. Front Microbiol. 2021;12:656306.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWest KH, et al. Non-native peptides capable of pan-activating the agr quorum sensing system across multiple specificity groups of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e. ACS Chem Biol. 2021;16(6):1070\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEisenbraun EL, Vulpis TD, Prosser BN, Horswill AR, Blackwell HE. Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities. J Am Chem Soc. 2024;146(23):15941\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDurham PG et al. Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant S\u003cem\u003etaphylococcus aureus\u003c/em\u003e biofilms, \u003cem\u003eBiofilm\u003c/em\u003e, vol. 3, p. 100049, 2021.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePai L, Patil S, Liu S, Wen F. A growing battlefield in the war against biofilm-induced antimicrobial resistance: insights from reviews on antibiotic resistance. Front Cell Infect Microbiol. 2023;13:1327069.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJo D-M et al. Green Medicine: Advancing Antimicrobial Solutions with Diverse Terrestrial and Marine Plant-Derived Compounds, \u003cem\u003eProcesses\u003c/em\u003e, vol. 12, no. 11, p. 2316, 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKošćak L, Lamovšek J, Đermić E, Prgomet I, Godena S. Microbial and plant-based compounds as alternatives for the control of phytopathogenic bacteria, \u003cem\u003eHorticulturae\u003c/em\u003e, vol. 9, no. 10, p. 1124, 2023.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, et al. Natural antimicrobials from plants: Recent advances and future prospects. Food Chem. 2024;432:137231.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNwafor IR et al. Plant-derived Bioactive Compounds and Their Mechanistic Roles in Combating Microbial Biofilms.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoozari M, Soltani S, Iranshahi M. Biologically active prenylated flavonoids from the genus \u003cem\u003eSophora\u003c/em\u003e and their structure\u0026ndash;activity relationship\u0026mdash;A review. Phytother Res. 2019;33(3):546\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEmami SA, Amin-Ar-Ramimeh E, Ahi A, Bolourian Kashy MR, Schneider B, Iranshahi M. Prenylated flavonoids and flavonostilbenes from Sophora pachycarpa. roots. Pharm Biol. 2007;45(6):453\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim D, Kim K-y. Antibacterial effect of sophoraflavanone G by destroying the cell wall of \u003cem\u003eEnterococcus faecium\u003c/em\u003e. J Appl Pharm Sci. 2020;10(9):059\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeng Z, et al. Antimicrobial activities of lavandulylated flavonoids in \u003cem\u003eSophora flavences\u003c/em\u003e against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e via membrane disruption. J Adv Res. 2024;57:197\u0026ndash;212.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan G et al. One Earth-One Health (OE-OH): Antibacterial Effects of Plant Flavonoids in Combination with Clinical Antibiotics with Various Mechanisms, \u003cem\u003eAntibiotics\u003c/em\u003e, vol. 14, no. 1, p. 8, 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFakhimi A, Iranshahi M, Emami SA, Amin-Ar-Ramimeh E, Zarrini G, Shahverdi AR. Sophoraflavanone G from \u003cem\u003eSophora pachycarpa\u003c/em\u003e enhanced the antibacterial activity of gentamycin against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Z f\u0026uuml;r Naturforschung C. 2006;61:9\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCha J-D, Jeong M-R, Jeong S-I, Lee K-Y. Antibacterial activity of sophoraflavanone G isolated from the roots of \u003cem\u003eSophora flavescens\u003c/em\u003e. J Microbiol Biotechnol. 2007;17(5):858\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHwang S-M et al. Antibacterial activity of Sophoraflavanone G isolated from the roots of \u003cem\u003eSophora flavescens\u003c/em\u003e and Red Ginseng Extract, 한국미생물학회 학술대회논문집, pp. 236\u0026ndash;236, 2013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao Y et al. Sophoraflavanone G: A review of the phytochemistry and pharmacology, \u003cem\u003eFitoterapia\u003c/em\u003e, vol. 177, p. 106080, 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShakeri A, Khakdan F, Soheili V, Sahebkar A, Rassam G, Asili J. Chemical composition, antibacterial activity, and cytotoxicity of essential oil from \u003cem\u003eNepeta ucrainica\u003c/em\u003e L. spp. \u003cem\u003ekopetdaghensis\u003c/em\u003e. Ind Crops Prod. 2014;58:315\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBashi DS, Dowom SA, Bazzaz BSF, Khanzadeh F, Soheili V, Mohammadpour A. Evaluation, prediction and optimization the ultrasound-assisted extraction method using response surface methodology: antioxidant and biological properties of \u003cem\u003eStachys parviflora\u003c/em\u003e L. Iran J basic Med Sci. 2016;19(5):529.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia F, Zhou Q, Li X, Zhou X. Total alkaloids of \u003cem\u003eSophora alopecuroides\u003c/em\u003e and matrine inhibit auto-inducer 2 in the biofilms of \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e. Microb Pathog. 2019;136:103698.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChan BC-L, et al. Quick identification of kuraridin, a noncytotoxic anti-MRSA (methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e) agent from \u003cem\u003eSophora flavescens\u003c/em\u003e using high-speed counter-current chromatography. J Chromatogr B. 2012;880:157\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsuchiya H, et al. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. J Ethnopharmacol. 1996;50(1):27\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFakhimi A, Iranshahi M, Emami SA, Amin-Ar-Ramimeh E, Zarrini G, Shahverdi AR. Sophoraflavanone G from \u003cem\u003eSophora pachycarpa\u003c/em\u003e enhanced the antibacterial activity of gentamycin against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Z f\u0026uuml;r Naturforschung C. 2006;61:11\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOh I, Yang W-Y, Chung S-C, Kim T-Y, Oh K-B, Shin J. In vitro sortase A inhibitory and antimicrobial activity of flavonoids isolated from the roots of \u003cem\u003eSophora flavescens\u003c/em\u003e. Arch Pharm Res. 2011;34:217\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan G, Guan Y, Yi H, Lai S, Sun Y, Cao S. Antibacterial activity and mechanism of plant flavonoids to gram-positive bacteria predicted from their lipophilicities. Sci Rep. 2021;11(1):10471.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWan C-X, Luo J-G, Ren X-P, Kong L-Y. Interconverting flavonostilbenes with antibacterial activity from \u003cem\u003eSophora alopecuroides\u003c/em\u003e, \u003cem\u003ePhytochemistry\u003c/em\u003e, vol. 116, pp. 290\u0026ndash;297, 2015.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAn J-X et al. Antibacterial Activities of Prenylated Flavonoids from Sophora flavences against \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv oryzae. J Agric Food Chem, 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsuchiya H, Iinuma M. Reduction of membrane fluidity by antibacterial sophoraflavanone G isolated from \u003cem\u003eSophora exigua\u003c/em\u003e, \u003cem\u003ePhytomedicine\u003c/em\u003e, vol. 7, no. 2, pp. 161\u0026ndash;165, 2000.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMirjani R, Rafii F, Sharifzadeh M, Amanlou M, Shahverdi AR. Post-antibiotic and post-antibiotic sub-MIC effects of gentamicin, sophoraflavanone G, and their combination against a clinical isolate of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Asian Biomed. 2010;4:821\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Z-L, Sun S-C, He J-M, Lan J-E, Gibbons S, Mu Q. Synergism of sophoraflavanone G with norfloxacin against effluxing antibiotic-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Int J Antimicrob Agents. 2020;56(3):106098.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSychrov\u0026aacute; A, Škovranov\u0026aacute; G, Čulenov\u0026aacute; M, Bittner S, Fialov\u0026aacute;. Prenylated flavonoids in topical infections and wound healing, \u003cem\u003eMolecules\u003c/em\u003e, vol. 27, no. 14, p. 4491, 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFan L, Liu Z, Zhang Z, Bai H. Antimicrobial Effects of \u003cem\u003eSophora flavescens\u003c/em\u003e Alkaloid s on Metronidazole-Resistant \u003cem\u003eGardnerella vaginalis\u003c/em\u003e in Planktonic and Biofilm Conditions. Curr Microbiol. 2023;80(8):263.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim CS et al. Antimicrobial effect of sophoraflavanone G isolated from \u003cem\u003eSophora flavescens\u003c/em\u003e against mutans streptococci, \u003cem\u003eAnaerobe\u003c/em\u003e, vol. 19, pp. 17\u0026ndash;21, 2013.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sophoraflavanone G, Biofilm, Pseudomonas aeruginosa, Staphylococcus epidermidis, Antimicrobial activity, Plant-derived compound","lastPublishedDoi":"10.21203/rs.3.rs-7299693/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7299693/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance, particularly among biofilm-forming pathogens, poses a serious threat to public health and drives the urgent need for new therapeutic agents. Sophoraflavanone G, a prenylated flavonoid derived from \u003cem\u003eSophora pachycarpa\u003c/em\u003e, has shown notable antimicrobial activity, especially against Gram-positive bacteria. In this study, Sophoraflavanone G was isolated and purified using chromatographic techniques and structurally confirmed via \u0026sup1;H-NMR. Its antibacterial and anti-biofilm properties were evaluated against several bacterial strains, including \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e PAO1 and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e DSMZ 3270. The compound exhibited potent inhibitory and bactericidal activity against all tested Gram-positive bacteria, with \u003cem\u003eListeria monocytogenes\u003c/em\u003e being the most sensitive (MIC\u0026thinsp;=\u0026thinsp;0.98 \u0026micro;g/mL). However, it showed no activity against \u003cem\u003eP. aeruginosa\u003c/em\u003e in planktonic form (MIC\u0026thinsp;\u0026gt;\u0026thinsp;1000 \u0026micro;g/mL). While it failed to inhibit \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm formation or enhance tobramycin penetration at low doses, a higher concentration (1 mg/mL) of Sophoraflavanone G significantly improved antibiotic penetration into the biofilm. In contrast, the compound demonstrated strong inhibitory, disruptive, and biofilm-penetrating effects against \u003cem\u003eS. epidermidis\u003c/em\u003e, with a clear dose-dependent response. These findings underscore the potential of Sophoraflavanone G as a candidate for managing Gram-positive and biofilm-associated infections, particularly those involving \u003cem\u003eS. epidermidis\u003c/em\u003e, while highlighting the need for further development to improve its efficacy against Gram-negative pathogens.\u003c/p\u003e","manuscriptTitle":"Antibacterial Evaluation of Sophoraflavanone G from Sophora pachycarpa Against Pseudomonas aeruginosa and Staphylococcus epidermidis Biofilm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 16:07:22","doi":"10.21203/rs.3.rs-7299693/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-22T09:45:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T10:10:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222249718747604826609195761042636410025","date":"2025-12-08T10:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116745054810072590819450020879499246888","date":"2025-12-08T06:08:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T16:37:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113385160831416156499259979060009538910","date":"2025-11-12T14:50:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T04:08:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-22T23:34:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-22T23:32:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T21:23:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2025-09-04T21:19:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f8953a6-9052-4c56-8bfb-eff00f003356","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-22T21:08:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 16:07:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7299693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7299693","identity":"rs-7299693","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}